Field of the Invention
The invention relates to a method for fast, simple, and highly sensitive isolation of viral nucleic acids.
Description of the Related Art
The examination of diagnostically relevant biological samples (serum, plasma, blood, spinal fluid, swab samples, abraded organ tissue, etc.) for the detection of infectious pathogens is increasingly gaining importance. Virus infections such as HIV, HCV, or HBV are continuing to spread more strongly worldwide. Furthermore, the massive occurrence of new types of virus diseases (e.g. bird flu) requires a fast and flexible reaction for sensitive diagnostic virus detection. New test methods on the basis of using sensitive amplification techniques such as Real Time PCR allow highly efficient virus detection, and are increasingly being used as diagnostic instruments. The step of molecular sample preparation in the form of isolation of the nucleic acids to be detected is increasingly gaining importance.
The sensitivity of the test system is decisively influenced by the process of isolation of the nucleic acid.
A number of extraction methods exist, which allow isolation of viral nucleic acids. U.S. Pat. No. 5,234,809 describes a process for isolation of nucleic acids from starting materials that contain nucleic acid, by means of incubation of the starting material with a chaotropic buffer and a solid phase that binds DNA. The solid phase consists of silicate mineral particles which are >50 nm. The chaotropic buffers implement both lysis of the starting material and binding of the nucleic acids to the solid phase. The method is well suited for isolating nucleic acids from small sample amounts, and finds its practical use particularly in the sector of the isolation of viral nucleic acids. However, it has been shown in practical use that the method described in U.S. Pat. No. 5,234,809 brings with it a number of disadvantages. The use of particulate systems for binding of the nucleic acids to be isolated is fundamentally very complicated in its handling. Furthermore, it has been shown that the use of buffers on the basis of the use of chaotropic salts is often not sufficient with regard to a required, highly sensitive virus detection.
Other methods describe chromatographic purification and separation of nucleic acids from a solution that has a high salt concentration and a high alcohol concentration.
The solution is again brought into contact with a carrier material for binding the nucleic acids. Subsequently, the carrier material is washed with known alcoholic washing buffers, and the bound nucleic acid is finally dissolved from the carrier material again, by means of water or a low-salt buffer.
The method implements efficient isolation and, if necessary, also separation of nucleic acids/nucleic acid mixtures, and is simple and quick in its implementation. The improvement as compared with U.S. Pat. No. 5,234,809 is achieved by means of the addition of an alcohol to the initial lysis buffer. This step allows an improvement in efficiency of the extraction process, which makes it possible to isolate even nucleic acid targets from a clinical sample, in sensitive manner.
Another method for separation and isolation of single-stranded and double-stranded nucleic acids is disclosed in EP 1 146 049 A2. The method is based on the treatment of a source that contains nucleic acids, using at least one mineral carrier material, in such a form that:                1. the single-stranded nucleic acid is isolated, in that the treatment conditions are adjusted by means of an aqueous mixture of chaotropic salts and low aliphatic alcohols, in such a manner that subsequently, the single-stranded nucleic acid is primarily adsorbed onto a mineral carrier, while the double-stranded nucleic acid is not adsorbed. The bound, single-stranded nucleic acid is subsequently washed and finally eluted from the carrier material by means of a low-salt buffer.        2. the double-stranded nucleic acid is isolated, in that the treatment conditions are adjusted by means of a mixture of substances that complex earth-alkali ions, without low aliphatic alcohols, in such a manner that subsequently, the double-stranded nucleic acid is primarily adsorbed onto a mineral carrier, while the single-stranded nucleic acid is not adsorbed. The bound, double-stranded nucleic acid is subsequently washed and finally eluted from the carrier material by means of a low-salt buffer.        3. the double-stranded nucleic acid is isolated in that the treatment conditions are adjusted by means of the presence of a sarcosinate but without low aliphatic alcohols, in such a manner that subsequently, the double-stranded nucleic acid is primarily adsorbed onto a mineral carrier, while the single-stranded nucleic acid is not adsorbed. The bound, double-stranded nucleic acid is subsequently washed and finally eluted from the carrier material by means of a low-salt buffer.        4. the double-stranded or single-stranded nucleic acid is isolated in that the treatment conditions are adjusted by means of an aqueous mixture of chaotropic salts and low aliphatic alcohols, in such a manner that both nucleic acid fractions adsorb onto a mineral carrier. Separation of the nucleic acids takes place by means of selective elution. In this connection, the double-stranded nucleic acid is dissolved from the carrier material by means of a solution having a reduced ion strength and low aliphatic alcohols. The remaining single-stranded nucleic acid is subsequently washed and finally dissolved from the carrier material by means of a low-salt buffer.        
Alternatively to this, the single-stranded nucleic acid can be selectively eluted from the carrier material by means of a solution that contains substances that complex earth-alkali ions and/or contains sarcosinates. The now remaining double-stranded nucleic acid is subsequently washed, once again, and finally dissolved from the carrier material by means of a low-salt buffer.
Again, the method is simple in its implementation, and can isolate DNA or RNA from biological samples. Although EP 1 146 049 A2 relates to the separation of nucleic acid mixtures, it becomes clear to a person skilled in the art, when reading, for example, dependent claim 4, that again, as described above, binding of nucleic acids to a mineral material is implemented by means of the combination of a salt solution with an alcohol. Again, the decisive factor for the step of binding of nucleic acids to mineral materials, in this connection, is the existence of an alcohol in the buffers used for the extraction process. Thus, the background art indicates that isolation of nucleic acids from biological samples using chaotropic salts alone works, but that the method only becomes really efficient by adding an alcohol (i.e. a combination of a solution that contains salt with an aliphatic alcohol).
In the European patent application EP 1 524 317 A1, a lysis buffer is described, among other things, that contains 5% Triton and no indication of an alcohol. In Example 1, a buffer is used that is composed of 6 M guanidine hydrochloride, 10 mM tris-HCl, and 20% Triton X-100. This buffer is added to the biological sample. Afterwards, the addition of proteinase K also takes place. Subsequently, incubation at 70° C. takes place. It is obvious that this buffer, in combination with the proteinase K, thus fulfills the function of a lysis buffer. After the lysis, another component is added to the batch (e.g. isopropanol, acetonitrile, DMSO, or methyl ethyl ketone), whereby this involves a non-acidic organic component miscible in water. In another example, N-methyl-2-pyrrolidone, or, alternatively to this, isopropanol is also used as a “non-acidic” organic component. As Example 3 explains, this also relates to the isolation of DNA by means of binding of the nucleic acid to magnetic particles. Here, too, the addition of N-methyl-2-pyrrolidone shows similar yields as the addition of isopropanol. Example 4 also uses a lysis buffer, whereby again, after lysis of the sample, the addition of a binding conditioner (gamma butyl lactone, propylene carbonate, or again, N-methyl-2-pyrrolidone) takes place. The detergent component that is listed in EP 1 524 317 A1 (Triton X-100) is merely a component of the lysis buffer. However, there is no indication at all that this component mediates binding of DNA or RNA. According to the patent specification, this is implemented by means of the additional addition of the non-acidic organic components according to the invention. It turns out that the lysis buffer is not able to implement isolation of nucleic acids. For the isolation of DNA or RNA, the listed organic acid must be added. The components listed in EP 1 524 317 A1 are furthermore also highly toxic (e.g. DMSO) and represent a significant hazard potential.